Abstract: Background and Objective: Out of a large number of plant species used for curing various ailments, there are certain species which are not used widely but occur immensely in major parts of the state. Folklore claims also support the uses of such species for curing various diseases. In the present study one of such species, Lantana camara (L. camara) have been selected to investigate the in vitro antifungal activities of the leaf extract in different solvents viz., acetone, chloroform, ethanol and methanol extracts of the selected plant Lantana camara. Methodology: Poisoned food technique was carried out to perform the antifungal activity of acetone, chloroform, ethanol and methanol extracts. Results: All the extracts of L. camara gave positive for all the phytoconstituents viz. alkaloids, carbohydrates, flavonoids, glycosides, phytosterols, phenols, proteins and amino acids, saponins and tannins. Among all the extracts ethanol and methanol extracts showed better results. Methanol leaf extract of L. camara showed broad antifungal activity against both the fungal strains. Terbinafine which was used as standard completely inhibited fungal growth of both Aspergillus flavus and Aspergillus niger. Conclusion: The phytochemical screening and efficient antifungal activity of Lantana camara from the present investigation revealed that the methanol leaf extracts of the selected plant have significant potential to inhibit the growth of pathogenic fungal strains than other leaf extracts.
Nature has been a source of therapeutic agents for thousands of years and a good number of modern drugs have been isolated from natural resources1. The presence of different phytochemicals in different parts of plants validates the ability of the plant to provide a source of natural medicines2. Plant extracts and their active phytochemical compounds have been used for antimicrobial activities and have significant remedial properties. Medicinal plants represent a rich source of secondary metabolites, many of which possess antimicrobial properties 3. A good perceptive of plants offers the potential of developing effective broad spectrum antibiotics. Medicinal plants represent an alternative cure in non-severe cases of infectious diseases4. Some Indian plants have been reported to show antimicrobial, antioxidant and anti-inflammatory properties5-7. Plants can act as a source for new effective antibiotics.
Lantana camara L. is a species of flowering plant of the family Verbenaceae. It is a small perennial shrub which can grow to around 2 m tall and form dense thickets in varied environments. The plant is native to American tropics8. It has been widely used in traditional medicine for the treatment of various disorders like cancer, tumors, malaria, tetanus, ulcers, high blood pressure, eczema, wound healing, eye infections, chicken pox, measles, asthma, rheumatism and fever9. It is an excellent source of various classes of bioactive natural products like steroids, flavonoids, oligosaccharides, triterpenoids, glycosides and naphthoquinones10-12.Various important phytomolecules such as oleanolic acid, lantanoside, linaroside, verbascoside, ursolic acid, camarinic acid, umuhengerin and phytol have been isolated from L. camara and their biological actions such as anticancer, antioxidant, hepatoprotective, leishmanicidal, antibacterial, nematicidal and antiulcer have been reported9,10.
Different plants are being used for medicinal purposes in many countries as a source of potent drugs13. The interest in present scientific research of L. camara was based on different evidences of its effective use for the curing many diseases. Present study revealed the presence of phytochemicals and in vitro anti-fungal properties of acetone, chloroform, ethanol and methanol extracts of the leaves of L. camara against Aspergillus flavus and Aspergillus niger fungal strains.
MATERIALS AND METHODS
Plant material: Fresh leaves of L. camara were collected from the Medicinal Plant Garden, School of Studies in Botany, Jiwaji University, Gwalior during 2015-2016. The plant was identified by the Institute of Ethnobiology Herbarium, Jiwaji University, Gwalior (India).
Processing of the plant material: To avoid contamination, leaves were washed with running tap water and then rinsed properly with distilled water. The shade dried leaves of L. camara were finely grinded using electric grinder and stored in air tight containers for future use. The powdered material (approx. 100 g) was extracted in soxhlet apparatus for 6 h in acetone, chloroform, ethanol and methanol (Merck, India). The extracts were then filtered through Whatmans No. 1 filter paper. The excess solvent in the extracts was removed by distillation and the concentrated extracts so obtained were further dried at a temperature not exceeding 100°C in water bath. The extracts were collected in air tight containers and stored at 4°C for further analysis.
Test microorganisms: The test microorganisms used in the investigation were Aspergillus flavus and Aspergillus niger. These fungal isolates were sub-cultured on a potato dextrose agar (PDA) for 72 h at 25°C.
Positive and negative control: Fluconazole (1mg mL1) and terbinafine (1 mg mL1) were used as positive control for the test of fungal strains. Petri plates without plant extract were used as negative control.
Assay for anti-fungal activity: The poisoned food technique was used for the determination of anti-fungal activity of L. camara leaf extracts in different solvents14. One petri plate containing potato dextrose agar (PDA) and standard anti-fungal was used as positive control. Petri plates without plant extract were used as negative control. The petri plates were incubated at 37°C for 7 days. Cultures were examined thrice weekly during the incubation period. Readings were taken by measuring mycelial growth zone of fungus in petri plates and percentage inhibition of fungal growth for each concentration of all extracts was determined as:
Statistical analysis: The data were analyzed by using Statistical Ezanova version 2. The data is presented as mean±standard error of two replicates.
Phytochemical screening: All the extracts of L. camara gave positive test when different reagents were used to test viz. alkaloids, carbohydrates, flavonoids, glycosides, phytosterols, phenols, proteins and amino acids, saponins and tannins (Table 1). Although some of the phytoconstituents were found in trace amount but there was presence of these phytoconstituents. Among all the extracts ethanol and methanol extracts showed better results.
Antifungal activity: The methanol leaf extract of L. camara showed broad antifungal activity against the tested microorganism at concentration of 3 mL (15%). Methanol leaf extract inhibited the fungal growth of Aspergillus flavus by 80.74 at 15% concentration (3 mL) while as acetone, chloroform and ethanol showed almost similar inhibition results against A. flavus i.e., 77.78% at the concentration of 15% (Table 2). Fluconazole inhibited fungal growth of A. flavus by 75.56% (Fig. 1 and 2).
Highest inhibition in fungal growth of A. niger was exhibited by methanol (79.07%) followed by acetone (76.74%) at 20% concentration. Chloroform and ethanol leaf extracts inhibited the fungal growth of A. niger by 48.06% (at 15% conc.) and 68.99 at 20% conc. (Fig. 2). Fluconazole inhibited fungal growth of A. niger by 52.71% (Table 3).
Preliminary phytochemical screening, antifungal activity and the percentage inhibition of the acetone, chloroform, ethanol and methanol extract of the leaves of L. camara L. against Aspergillus flavus and Aspergillus niger fungal strains have been evaluated in the present research work. The in vitro antimicrobial activity evaluation of different plant extracts was a first step towards the development of new potential drugs. The acetone, chloroform, ethanol and methanol leaf extracts of Lantana camara L. tested on Aspergillus flavus and Aspergillus niger fungal strains showed a dose dependent antifungal activity. This agrees with several reports in which similar observations were made15. The antifungal activity of the leaf extracts at higher doses on Aspergillus flavus and Aspergillus niger could be attributed to the presence of some bioactive components in the extracts.
Generally the methanol leaf extract showed a higher growth inhibition on all the organisms used in this study. The effect of methanolic leaf extract is significantly higher on Aspergillus flavus and Aspergillus niger as compared to other solvents used. This finding agrees with the report of Pranoothi and co workers16 in which the whole plant extract of Leucas indica was found to significantly inhibit the growth of Candida rogasa, Rhizopus oryzae, Aspergillus niger and Candida albicans. The report of Satish et al.17 revealed that methanol plant extract was found to exhibit marked antifungal activity than ethanol, chloroform, benzene and petroleum ether. This study also agrees with the report of Sailaja18 in which methanolic extracts of Lantana camara L. and Oscimum basilicum L., showed highest MIC value of 0.7 mg mL1 against Aspergillus niger. The results of the present study did not agree with the report of Pandithurai et al.19 in which the maximum activity was recorded from chloroform extract against the non dermatophytic fungi Aspergillus flavus (98.83%)19. The study of Ashraf et al.20 on antifungal activity of methanol, chloroform and aqueous extracts of Origanum vulgare against Aspergillus flavus, Aspergillus niger and Aspergillus pterus showed high susceptibility of fungal strains to chloroform extract which is in disagreement with the present study. This study showed methanol extract had higher antifungal activity against Aspergillus flavus and Aspergillus niger as compared to the other extracts of the same plant. The MIC showing Aspergillus flavus and Aspergillus niger being more susceptible to the methanolic leaf extract when compared with did not agree with Linthoingambi and Singh21, who reported that the petroleum ether leaf extract had highest antifungal activity followed by methanol and chloroform extracts.
With the spread of antibiotic resistance and noticeable challenges with medicinal systems in the treatment of many infectious diseases, such plants should be considered to obtain their all possible antimicrobial benefits. The biologically active compounds having antimicrobial potential must be extracted and then identified. The tolerable level and toxic effects of such compounds should be properly investigated. This study provided a scientific validation to this medicinal plant to be used as antimicrobial drug. This study further suggested the isolation and identification of the phytochemicals and active compounds in the plant.
The extracts of the plant part used showed prominent antifungal activity against Aspergillus niger and Aspergillus flavus which are severe pathogens. Thus, the use of this plant in the treatment of pathogenic diseases associated with the infection of these pathogens is validated, scientifically supported by the results obtained in this study. Therefore, further efficacy and safety studies are encouraged onthis medicinal weed as alternative and effective in clinical practice.
The present finding shows that different solvent extracts of a plant species may have different spectra of antifungal activity that can be explained by the solubility or insolubility of the active compound(s) in the solvent used for extraction. This study adequately justifies the ethno-medical use of this plant in the management of some fungal diseases. This study further suggests the researchers to isolate and identify the phytochemicals and active compounds in the plant.
Authors are thankful to the Institute of Ethnobiology and School of Studies in Botany, Jiwaji University, Gwalior (India) for providing the needed facilities to carry out this research.